Combination therapy for glycaemic control

ABSTRACT

The present invention relates to method of treatment, in particular to a method for the treatment of diabetes mellitus, especially non-insulin dependent diabetes mellitus (NIDDM) or Type 2 diabetes and conditions associated with diabetes mellitus the predi,betic state and/or obesity and to compositions for use in s ch method.

FIELD OF THE INVENTION

This invention relates to a therapy for glycaemic control, in particularto a method for the treatment of diabetes mellitus, especiallynon-insulin dependent diabetes mellitus (NIDDM) or Type 2 diabetes andconditions associated with diabetes mellitus, the prediabetic stateand/or obesity and to compositions for use in such method.

BACKGROUND ART

Glycaemic control is therapeutically important in the treatment ofconditions such as diabetes mellitus and related conditions. Clinicaldiabetes may be divided into four general subclasses, including (1) type1 or insulin-dependent diabetes mellitus (IDDM) (caused by beta celldestruction and characterized by absolute insulin deficiency), (2) type2 or non-insulin-dependent diabetes (NIDDM) (characterized by insulinresistance and relative insulin deficiency, (3) other specific types ofdiabetes (associated with various identifiable clinical conditions orsyndromes such as genetic defects of β-cell function e.g. maturity-onsetdiabetes of the young [MODY] types 1-3 and point mutations inmitochondrial DNA), and (4) gestational diabetes mellitus.

Type 2 diabetes is by far the most common form of the disease, is foundin over 90% of the diabetic patient population. These patients retain asignificant level of endogenous insulin secretory capacity. However,insulin levels are low relative to the magnitude of insulin resistanceand ambient glucose levels. Type 2 patients are not dependent on insulinfor immediate survival and ketosis rarely develops, except underconditions of great physical stress. Nevertheless, these patients mayrequire insulin therapy to control hyperlgycemia. Type 2 diabetestypically appears after the age of 40 years, has a high rate of geneticpenetrance unrelated to specific immune response (HLA) genes, and isassociated with obesity.

In addition to these clinical categories, further conditions, namelyimpaired glucose tolerance and impaired fasting glucose, refer to ametabolic state intermediate between normal glucose homeostasis andovert diabetes (under fed and fasting conditions, respectively). Theseconditions significantly increase the later risk of diabetes mellitusand may in some instances be part of its natural history.

A further related condition is Impaired Glucose Metabolism (IGM) whichis defined by blood glucose levels that are above the normal range butare not high enough to meet the diagnostic criteria for type 2 diabetesmellitus. The incidence of IGM varies from country to country, butusually occurs 2-3 time more frequently than overt diabetes. Amongsubjects with IGM, about 58% have Impaired Glucose tolerance (IGT),another 29% have impaired fasting glucose (IFG), and 13% have bothabnormalities (IFG/IGT).

Many of the available treatments for type 2 diabetes, which have notchanged substantially in many years, have recognized limitations forexample they may have unwanted side effects, low efficacy or suffer fromefficacy loss over time during chronic treatment.

Increasing the plasma level of insulin by administration ofsulfonylureas (e.g. tolbutamide and glipizide) or meglitinide, whichstimulate the pancreatic (β-cells to secrete more insulin, and/or byinjection of insulin when sulfonylureas or meglitinide becomeineffective, can result in insulin concentrations high enough tostimulate the very insulin-resistant tissues. However, dangerously lowlevels of plasma glucose can result from administration of insulin orinsulin secretagogues (sulfonylureas or meglitinide), and an increasedlevel of insulin resistance due to the even higher plasma insulin levelscan occur. Alpha glucosidase inhibitor antihyperglycaemic agents (oralpha glucosidase inhibitors) and biguanide antihyperglycaemic agents(or biguanides) which increase insulin sensitivity resulting in somecorrection of hyperglycemia, are commonly used in the treatment of type2 diabetes. Acarbose, voglibose, emiglitate and miglitol are examples ofalpha glucosidase inhibitors. 1,1-Dimethylbiguanidine (or metformin) andphenformin are particular examples of biguanides, metformin has fewerside effects than phenformin.

The glitazones (i.e. 5-benzylthiazolidine-2,4-diones) are a morerecently described class of compounds with potential for amelioratingmany symptoms of type 2 diabetes. These agents substantially increaseinsulin sensitivity in muscle, liver and adipose tissue in severalanimal models of type 2 diabetes resulting in partial or completecorrection of the elevated plasma levels of glucose without occurrenceof hypoglycemia. The glitazones that are currently marketed are agonistsof the peroxisome proliferator activated receptor (PPAR), primarily thePPAR-gamma subtype. PPAR-gamma agonism is generally believed to beresponsible for the improved insulin sensititization that is observedwith the glitazones. Newer PPAR agonists that are being tested fortreatment of Type 2 diabetes are agonists of the alpha, gamma or deltasubtype, or a combination of these, and in many cases are chemicallydifferent from the glitazones. Side effects (e.g. liver toxicity) haveoccurred with some of the glitazones, such as troglitazone.

New approaches to the treatment of type 2 diabetes that have beenrecently introduced or are still under development include treatmentwith alpha-glucosidase inhibitors (e.g. acarbose) and protein tyrosinephosphatase-1B(PTP-1B) inhibitors.

Insulin secretagogues are compounds that promote increased secretion ofinsulin by the pancreatic beta cells. The sulphonylureas are well knownexamples of insulin secretagogues. The sulphonylureas act ashypoglycaemic agents and are used in the treatment of Type 2 diabetes.Examples of sulphonylureas include glibenclamide (or glyburide),glipizide, gliclazide, glimepiride, tolazamide and tolbutamide.

European Patent Application 0306228 discloses certain thiazolidinedionederivatives disclosed as having antihyperglycaemic and hypolipidaemicactivity, for example 5-[4-[2-(N-methyl-N-(2-pyridyl)amino)ethoxy]benzyl]thiazolidine-2,4-dione (rosiglitazone). WO 094/05659discloses certain salts of this compound including the maleate saltthereof. 5-[4-[2-(N-Methyl-N-(2-pyridyl)amino)ethoxy]benzyl]thiazolidine-2,4-dione is an example of a class ofantihyperglycaemic agents known as ‘insulin sensitisers’. In particularthis compound is a thiazolidinedione insulin sensitiser.5-[4-[2-(N-Methyl-N-(2-pyridyl)amino)ethoxy]-benzyl]thiazolidine-2,4-dioneis also a peroxisome proliferator-activated receptor (PPARy) agonistinsulin sensitiser.

European Patent Applications 0008203, 0139421, 0032128, 0428312,0489663, 0155845, 0257781, 0208420, 0177353, 0319189, 0332331, 0332332,0528734 and 0508740; International Patent Applications WO 92/18501, WO93/02079 and WO 93/22445 and U.S. Pat. Nos. 5,104,888 and 5,478,852,also disclose certain thiazolidinedione insulin sensitisers.

Another series of compounds generally recognised as having insulinsensitiser activity are those typified by the compounds disclosed inInternational Patent Applications WO 93/21166 and WO 94/01420. Thesecompounds are herein referred to as “acyclic insulin sensitisers”. Otherexamples of acyclic insulin sensitisers are disclosed in U.S. Pat. No.5,232,945 and International Patent Applications WO 92/03425 and WO91/19702. Examples of other insulin sensitisers are disclosed inEuropean Patent Application 0533933, Japanese Patent Application05271204 and U.S. Pat. No. 5,264,451.

Dipeptidyl peptidase IV (DP IV) is a serine protease which cleavesN-terminal dipeptides from a peptide chain containing, preferably, aproline residue in the penultimate position. Although the biologicalrole of DP IV in mammalian systems has not been completely established,it is believed to play an important role in neuropeptide metabolism,T-cell activation, attachment of cancer cells to the endothelium and theentry of HIV into lymphoid cells.

Likewise, it has been discovered that DP IV is responsible forinactivating glucagon-like peptide-1 (GLP-1) and glucose-dependentinsulinotropic peptide also known as gastric-inhibitory peptide (GIP).Since GLP-1 is a major stimulator of pancreatic insulin secretion andhas direct beneficial effects on glucose disposal, in WO 97/40832 andU.S. Pat. No. 6,303,661 inhibition of DP IV and DP IV-like enzymeactivity was shown to represent an attractive approach e.g. for treatingnon-insulin-dependent diabetes mellitus (NIDDM).

It is known that DP IV inhibitors may be useful for the treatment ofimpaired glucose tolerance and diabetes mellitus (International PatentApplication WO 99/61431, Pederson R. A. et al, Diabetes. 1998 August;47(8):1253-8 and Pauly R. P. et al, Metabolism 1999 March; 48(3):385-9).

WO 99/61431 discloses DP IV inhibitors comprising an amino acid residueand a thiazolidine or pyrrolidine group, and salts thereof, especiallyL-threo-isoleucyl thiazolidine, L-allo -isoleucyl thiazolidine,L-threo-isoleucyl pyrrolidine, L-allo-isoleucyl thiazolidine,L-allo-isoleucyl pyrrolidine, and pharmaceutically acceptable saltsthereof. WO 03/072556 discloses the DP IV inhibitors glutaminylthiazolidine and glutaminyl pyrrolidine and pharmaceutically acceptablesalts thereof.

It is the object of the present invention to provide new therapies forglycaemic control for example in the treatment of diabetes mellitus,especially non-insulin dependent diabetes (NIDDM) or Type 2 diabetes,conditions associated with diabetes mellitus, the pre-diabetic stateand/or obesity, which may exhibit greater efficiency and/or safety. Inparticular the present invention provides the use of combinations of theDP IV-inhibitors glutaminyl thiazolidine and glutaminyl pyrrolidine andother antidiabetic agents for glycaemic control, for example in thetreatment of diabetes mellitus, especially non-insulin dependentdiabetes (NIDDM) or Type 2 diabetes, conditions associated with diabetesmellitus, the pre-diabetic state and/or obesity.

SUMMARY OF THE INVENTION

The present invention provides a method for glycaemic control in amammal, such as a human, which method comprises administering aneffective amount of glutaminyl thiazolidine or glutaminyl pyrrolidine,or a pharmaceutically acceptable salt thereof, and another antidiabeticagent, to a mammal in need thereof.

The invention also provides the use of glutaminyl thiazolidine orglutaminyl pyrrolidine, or a pharmaceutically acceptable salt thereof,and another antidiabetic agent for glycaemic control.

The invention also provides the use of glutaminyl thiazolidine orglutaminyl pyrrolidine, or a pharmaceutically acceptable salt thereof,in the manufacture of a medicament for use in combination with anotherantidiabetic agent, for glycaemic control.

Glutaminyl thiazolidine and glutaminyl pyrrolidine have the followingstructure:

wherein for glutaminyl thiazolidine X=S and for glutaminyl pyrrolidineX=CH₂.

These compounds are hereinafter referred to as compounds of formula (I).

The combinations described above are of particular use for the treatmentof diabetes mellitus, especially Type 2 diabetes, and conditionsassociated with diabetes mellitus, the prediabetic state and/or obesity.In particular the treatment of Type 2 diabetes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 plots the blood glucose level over time for placebo, and threeadministered levels of glutaminyl pyrrolidine.

FIG. 2 plots the blood glucose level over time for placebo, and threeadministered levels of glutaminyl thiazolidine.

FIG. 3 is a chemical drawing of glutaminyl thiazolidine.

FIG. 4 is a chemical drawing of glutarninyl pyrrolidine.

FIG. 5 is a plot of the counts per second over time of glutaminylthiazolidine and pyroglutamic acid thiazolidine.

FIG. 6 shows the glucose AUC for various administered compositions.

FIG. 7 shows the glucose AUC for various administered compositions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for glycaemic control in amammal, such as a human, which method comprises administering aneffective amount of glutaminyl thiazolidine or glutaminyl pyrrolidine,or a pharmaceutically acceptable salt thereof, and another antidiabeticagent, to a mammal in need thereof.

The combinations are of particular use for the treatment of diabetesmellitus, especially Type 2 diabetes, and conditions associated withdiabetes mellitus, the prediabetic state and/or obesity. In particularthe treatment of Type 2 diabetes.

Such combinations provide a particularly beneficial effect on glycaemniccontrol and preferably provide improved blood glucose regulation withoutintroducing unacceptable side-effects

The present invention also provides a method for the treatment ofdiabetes mellitus, especially Type 2 diabetes, and conditions associatedwith diabetes mellitus, the prediabetic state and/or obesity, inparticular the treatment of Type 2 diabetes, in a mammal, such as ahuman, which method comprises administering an effective amount ofglutaminyl thiazolidine or glutaminyl pyrrolidine, or a pharmaceuticallyacceptable salt thereof, and another antidiabetic agent, to a mammal inneed thereof.

The invention also provides the use of glutaminyl thiazolidine orglutaminyl pyrrolidine, or a pharmaceutically acceptable salt thereof,and another antidiabetic agent for the treatment of diabetes mellitus,especially Type 2 diabetes, and conditions associated with diabetesmellitus, the prediabetic state and/or obesity, in particular thetreatment of Type 2 diabetes.

The invention also provides the use of glutaminyl thiazolidine orglutaminyl pyrrolidine, or a pharmaceutically acceptable salt thereof,in the manufacture of a medicament for use in combination with anotherantidiabetic agent, for the treatment of diabetes mellitus, especiallyType 2 diabetes, and conditions associated with diabetes mellitus, theprediabetic state and/or obesity, in particular the treatment of Type 2diabetes.

The compound of formula (I) and the other antidiabetic agent may beco-administered or administered sequentially or separately.

Co-administration includes administration of a formulation whichincludes both the compound of formula (I), or a pharmaceuticallyacceptable salt thereof and the other antidiabetic agent, or theessentially simultaneous administration of separate formulations of eachagent. Where the pharmacological profiles of the compound of formula(I), or a pharmaceutically acceptable salt thereof, and the otherantidiabetic agent allow it, coadministration of the two agents ispreferred.

The invention also provides the use of glutaminyl thiazolidine orglutaminyl pyrrolidine, or a pharmaceutically acceptable salt thereof,and another antidiabetic agent, in the manufacture of a medicament forglycaemic control.

The invention also provides the use of glutaminyl thiazolidine orglutaminyl pyrrolidine, or a pharmaceutically acceptable salt thereof,and another antidiabetic agent, in the manufacture of a medicament forthe treatment of diabetes mellitus, especially Type 2 diabetes, andconditions associated with diabetes mellitus, the prediabetic stateand/or obesity, in particular the treatment of Type 2 diabetes.

The invention also provides a pharmaceutical composition comprisingglutaminyl thiazolidine or glutaminyl pyrrolidine, or a pharmaceuticallyacceptable salt thereof, and another antidiabetic agent, and apharmaceutically acceptable carrier. The invention also encompasses theuse of such compositions in the methods described above.

The present invention includes the use of compounds of formula (I) andpharmaceutically acceptable salts thereof, according to any one of theembodiments of the present invention in combination with:

-   -   insulin sensitizers selected from the group consisting of PPAR        agonists, biguanides, and protein tyrosin phosphatase-1B (TP-1B)        inhibitors;    -   insulin and insulin mimetics;    -   sulfonylureas and other insulin secretagogues;    -   α-glucosidase inhibitors;    -   glucagon receptor agonists;    -   GLP-1; GLP-1 mimetics, e.g. NN-2211 (liraglutide from Novo        Nordisk), and GLP-1 receptor agonists;    -   GLP-2; GLP-2 mimetics, e.g. ALX-0600 (teduglutide from NPS        Allelix Corp.) and GLP-2 receptor agonists;    -   exendin-4 and exendin-4 mimetics, e.g. exenatide (AC-2993,        synthetic exendin-4 from Amylin/Eli Lilly);    -   GIP, GIP mimetics, and GIP receptor agonists;    -   PACAP, PACAP mimetics, and PACAP receptor 3 agonists;    -   cholesterol lowering agents selected from the group consisting        of HMG-CoA reductase inhibitors, sequestrants, nicotinyl        alkohol, nicotinic acid and salts thereof, PPARα agonists,        PPARα/γ dual agonists, inhibitors of cholesterol absorption,        acyl CoA:cholesterol acyltransferase inhibitors, and        antioxidants; and    -   PPARδ agonists;        and optionally other agents for example:    -   antiobesity compounds;    -   an ileal bile acid transporter inhibitor; and    -   anti-inflammatory agents.

Suitably, the other antidiabetic agent comprises one or more, generallyone or two, and especially one, of an alpha glucosidase inhibitor, abiguanide, an insulin secretagogue or an insulin sensitiser.

A further suitable antidiabetic agent is insulin.

A suitable alpha glucosidase inhibitor is acarbose.

Other suitable alpha glucosidase inhibitors are emiglitate and miglitol.A further suitable alpha glucosidase inhibitor is voglibose.

Suitable biguanides include metformin, buformin or phenformin,especially metformin.

Suitable insulin secretagogues include sulphonylureas.

Suitable sulphonylureas include glibenclamide, glipizide, gliclazide,glimepiride, tolazamide and tolbutamide. Further sulphonylureas includeacetohexamide, carbutamide, chlorpropamide, glibornuride, gliquidone,glisentide, glisolamide, glisoxepide, glyclopyamide and glycylamide.Also included is the sulphonylurea glipentide.

A further suitable insulin secretagogue is repaglinide. An additionalinsulin secretagogue is nateglinide.

Insulin sensitisers include PPARy agonist insulin sensitisers includingthe compounds disclosed in WO 97/31907 and especially2-(1-carboxy-2-{4-{2-(5-methyl-2-phenyl-oxazol-4-yl)ethoxy]phenylethylamino)benzoic acid methyl ester and 2(S)-(2-benzolyphenylamino)-3-{4-[2-(5-methyl-2-phenyl-oxazol-4-yl)ethoxy]phenyl}propionicacid.

Insulin sensitisers also include thiazolidinedione insulin sensitisers.

Other suitable thiazolidinedione insulin sensitisers include(+)-5-[[4-[(3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)methoxy]phenyl]methyl]-2,4-thiazolidinedione(or troglitazone),5-[4-[(1-methylcyclohexyl)methoxy]benzyl]thiazolidine-2,4-dione (orciglitazone),5-[4-[2-(5-ethylpyridin-2-yl)ethoxy]benzyl]thiazolidine-2,4-dione (orpioglitazone) or5-[(2-benzyl-2,3-dihydrobenzopyran)-5-ylmethyl)thiazolidine-2,4-dione(or englitazone).

Particular thiazolidinedione insulin sensitisers are5-[4-[2-(5-ethylpyridin-2-yl) ethoxy]benzyl]thiazolidine-2,4-dione (orpioglitazone) and(+)-5-[[4-[(3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)methoxy]phenyl]methyl]-2,4-thiazolidinedione(or troglitazone).

A preferred thiazolidinedione insulin sensitiser is5-[4-[2-(N-methyl-N-(2-pyridyl)amino)ethoxy]benzyl]thiazolidine-2,4-dione (or rosiglitazone) and saltsthereof.

Further antidiabetic agents include other inhibitors of DP IV.Particular DP IV-inhibitors include the specific examples disclosed inWO 99/61431, such as L-threo-isoleucyl pyrrolidide, L-allo-isoleucylthiazolidide, L-alloisoleucyl pyrrolidide and salts thereof. Aparticular DP IV-inhibitor is isoleucine thiazolidide and salts thereof.

Further DP IV-inhibitors include valine pyrrolidide (Novo Nordisk),NVP-DPP728A(1-[[[2-[{5-cyanopyridin-2-yl}amino]ethyl]amino]acetyl]-2-cyano-(S)-pyrrolidine)(Novartis) as disclosed by Hughes et al., Biochemistry, 38 (36),11597-11603, 1999, LAF-237 (1-[(3-hydroxy-adamant-1-ylamino)acetyl]pyrrolidine-2(S)-carbonitrile); disclosed by Hughes et al.,Meeting of the American Diabetes Association 2002, Abstract no. 272 or(Novartis), TSL-225(tryptophyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), disclosedby Yamada et.al., Bioorg. & Med. Chem. Lett. 8 (1998), 1537-1540,2-cyanopyrrolidides and 4-cyanopyrrolidides as disclosed by Asworth etal., Bioorg. & Med. Chem. Lett., 6, No. 22, pp 1163-1166 and 2745-2748(1996), FE-999011([(2S)-1-([2′S]-2′-amino-3′,3′dimethylbutanoyl)pyrrolidine-2-carbonitrile]),disclosed by Sudre et al., Diabetes 51 (5), pp 1461-1469 (2002)(Ferring) and the compounds disclosed in WO 01/34594 (Guilford),employing dosages as set out in the above references.

For the avoidance of doubt, the examples disclosed in each of the abovementioned publications are specifically incorporated herein by referencein their entirety, as individually disclosed compounds, especiallyconcerning their structure, their definition, uses and their production.

Preferred embodiments of the present invention comprise the use ofcompounds of formula (I), or pharmaceutically acceptable salts thereof,according to any one of the embodiments of the present invention:

-   in combination with acarbose, or-   in combination with metfomin; or-   in combination with acarbose and metformin; or-   in combination with an insulin sensitizer, e.g. a PPARy agonist    insulin sensitiser.

The use of a compound of formula (I), or a pharmaceutically acceptablesalt thereof, in particular glutaminyl thiazolidine hydrochloride, incombination with metformin e.g. for the treatment of diabetes mellitus,conditions associated with diabetes mellitus and conditions associatedwith the pre-diabetic state, is especially preferred according to thepresent invention. The compound of formula (I), or a pharmaceuticallyacceptable salt thereof, and metformin are preferably co-administered.

The further preferred aspect of the invention is a pharmaceuticalcomposition comprising glutaminyl thiazolidine or glutaminylpyrrolidine, or a pharmaceutically acceptable salt thereof, inparticular glutaminyl thiazolidine hydrochloride, and metformin, and apharmaceutically acceptable carrier. The pharmaceutical formulation ispreferably adapted for oral administration and in particular is in unitdoes form adapted for administration once, twice or three times,preferably twice or three times, a day.

The use of a compound of formula (I), or a pharmaceutically acceptablesalt thereof, in particular glutaminyl thiazolidine hydrochloride, incombination with an insulin sensitiser e.g. a PPARy agonist insulinsensitiser represents a further preferred aspect of the invention.Particular insulin sensitisers include the glitazones e.g. troglitazone,ciglitazone, pioglitazone, englitazone and rosiglitazone, in particularrosiglitazone.

It will be understood that the compounds of formula (I), orpharmaceutically acceptable salts thereof, and the other antidiabeticagents are each administered in a pharmaceutically acceptable form,including pharmaceutically acceptable derivatives such aspharmaceutically acceptable salts, esters and solvates thereof, asappropriate of the relevant pharmaceutically active agent. In certaininstances herein the names used for the other antidiabetic agent mayrelate to a particular pharmaceutical form of the relevant active agent.It will be understood that the use of all pharmaceutically acceptableforms of the active agents per se is encompassed by this invention.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof, possess several unexpected characteristics compared to other DPIV-inhibitors already known in the art, which may provide them withcertain advantages when administered in combination with otherantidiabetic agents according to the invention. These characteristicsinclude, for example:

-   -   no activity against non-DP IV and non-DP IV-like enzymes, e.g.        DP I, prolyl oligopeptidase, prolidase (see example 12);    -   high stability in isolated human plasma in vitro (see example        13);    -   a completely new and controllable mechanism of        inactivation/metabolism of the glutamine moiety to the        respective pyroglutaminyl compound in vivo, resulting in a        shorter half-life than other DP IV inhibitors (see example 8);        and    -   a presumably non-liver dependent half-life in vivo.

Pharmaceutically acceptable salts of the compounds of formula (I)include acid addition salts, i.e. where the amino acid basic side chainis protonated with an inorganic or organic acid. Representative organicor inorganic acids include hydrochloric, hydrobromic, perchloric,sulfuric, nitric, phosphoric, acetic, propionic, glycolic, lactic,succinic, maleic, fumaric, malic, tartaric, citric, benzoic, mandelic,methanesulfonic, hydroxyethanesulfonic, benzenesulfonic, oxalic, pamoic,2-naphthalenesulfonic, p-toulenesulfonic, cyclohexanesulfamic,salicylic, saccharinic, trifluoroacetic, sulfinic and3,5-di-tert-butylbenzoic acid. The use of all pharmaceuticallyacceptable acid addition salt forms of the compounds of formula (I) isembraced by the scope of this invention.

Preferred acid addition salts of the compounds of formula (I) are thefumarate, benzoate, maleinate, oxalate, 3,5-di-tertiary-butylbenzoate,salicylate, acetate and hydrochloride salts (see example 14). The mostpreferred acid addition salt of the compounds of formula (I) is thehydrochloride salt. The preferred compound of formula (I) beingglutaminyl thiazolidine hydrochloride.

For the avoidance of doubt whenever a compound of formula (I) isreferred to in the context of the present invention it is to beunderstood that reference is being made to both the free base and thecorresponding salts, provided such is possible or appropriate under thecircumstances.

The present invention further includes within its scope the use ofprodrugs of the compounds of formula (I). In general, such prodrugs willbe functional derivatives of the compounds which are readily convertiblein vivo into the desired therapeutically active compound. Thus, in thesecases, the methods of treatment of the present invention, the term“administering” shall encompass the treatment of the various disordersdescribed with prodrug versions of the compounds of formula (I) whichconverts to the specified compound in vivo after administration to thesubject. Procedures for the selection and preparation of suitableprodrug derivatives are described, for example, in “Design of Prodrugs”,ed. H. Bundgaard, Elsevier, 1985. Specific prodrugs are described inpatent applications DE 198 28 113, DE 198 28 114, WO 99/67228 and WO99/67279.

Where the compounds of formula (I) have at least one chiral center, theymay accordingly exist as enantiomers. In the case of compounds, e.g.prodrugs, which possess two or more chiral centers, they mayadditionally exist as diastereomers. It is to be understood that allsuch isomers and mixtures thereof are encompassed within the scope ofthe present invention.

Where the processes for the preparation of the compounds of formula (I)give rise to mixture of stereoisomers, these isomers may be separated byconventional techniques such as preparative chromatography. Thecompounds may be prepared in racemic form, or individual enantiomers maybe prepared either by enantiospecific synthesis or by resolution. Thecompounds may, for example, be resolved into their componentsenantiomers by standard techniques, such as the formation ofdiastereomeric pairs by salt formation with an optically active acid,such as (−)-di-p-toluoyl-d-tartaric acid and/or(+)-di-p-toluoyl-1-tartaric acid followed by fractional crystallizationand regeneration of the free base. The compounds may also resolved byformation of diastereomeric esters or amides, followed bychromatographic separation and removal of the chiral auxiliary.Alternatively, the compounds may be resolved using a chiral HPLC column.

Where the compounds of formula (I) are preferably haveL-alpha-glutylamine derivatives.

During any of the processes for preparation of the compounds of formula(I), it may be necessary and/or desirable to protect sensitive orreactive groups on any of the molecules concerned. This may be achievedby means of conventional protecting groups, such as those described inProtective Groups in Organic Chemistry, ed. J. F. W. McOmie, PlenumPress, 1973; and T. W. Greene & P. G. M. Wuts, Protective Groups inOrganic Synthesis, John Wiley & Sons, 1991. The protecting groups may beremoved at a convenient subsequent stage using conventional methodsknown from the art.

Furthermore, some of the crystalline forms of the compounds of formula(I) may exist as polymorphs and as such are included in the presentinvention. In addition, some of the compounds may form solvates withwater (i.e. hydrates) or common organic solvents, and such solvates arealso intended to be encompassed within the scope of this invention.

The compounds of formula (I), and pharmaceutically acceptable saltsthereof, can also be obtained in the form of their hydrates, or includeother solvents used for their crystallization.

As indicated above, the compounds of formula (I), and pharmaceuticallyacceptable salts thereof, are useful in inhibiting DP IV and DP IV-likeenzyme activity. The ability of the compounds of formula (I), andpharmaceutically acceptable salts thereof, to inhibit DP IV and DPIV-like enzyme activity may be demonstrated employing the DP IV activityassay for determination of the K_(i)-values in vitro and in humanplasma, as described in examples 4 and 5. The K_(i)-values of thecompounds of the present invention were determined for glutaminylthiazolidine as K_(i)=3.12*10⁻⁷ M±5.11*10⁻¹⁰ M and for glutaminylpyrrolidine as K_(i)=1.30*10⁻⁶ M±8.49*10⁻⁸ M against porcine kidney DPIV. The K_(i)-values of the compounds of the present invention weredetermined for glutaminyl thiazolidine as K_(i)=4.03*10⁻⁷ M±2.19*10⁻¹⁰ Mafter 5 min 5.13*10⁻⁷ M±1.26*10⁻⁸ M after 22 hours pre-incubation, andfor glutaminyl pyrrolidine as K_(i)=1.30*10⁻⁶ M±4.89*10⁻⁸ M after 5 minand 1.36*10⁻⁶ M±3.21*10⁻⁸ M after 22 hours pre-incubation in humanplasma.

The ability of the compounds of formula (I), and pharmaceuticallyacceptable salts thereof, to inhibit DP IV in vivo may be demonstratedby oral or intravasal administration to Wistar rats, as described inexample 9. The compounds inhibit DP IV activity in vivo after both, oraland intravasal administration to Wistar rats.

The compounds of formula (I), and pharmaceutically acceptable saltsthereof, are able to inhibit DP IV in vivo.

The compounds of formula (I) and pharmaceutically acceptable salt,thereof improve glucose tolerance by lowering elevated blood glucoselevels in response to an oral glucose challenge and, therefore, areuseful in treating non-insulin-dependent diabetes mellitus. The abilityof the compounds of formula (I), and pharmaceutically acceptable saltstherof, to improve glucose tolerance in response to an oral glucosechallenge, may be measured in diabetic Zucker rats. The method isdescribed in examples 6 and 7. Oral administration of 5 mg/kg b.w., 15mg/kg and 50 mg/kg b.w. glutaminyl thiazolidine or glutaminylpyrrolidine resulted in a dose dependent lowering of elevated bloodglucose levels and thereby in an improvement of glucose tolerance indiabetic Zucker rats.

Surprisingly, the compounds of formula (I), and pharmaceuticallyacceptable salts thereof, are degraded in vivo in a controllable mannerfollowing administration to a mammal. The ability of the compounds offormula (I), and pharmaceutically acceptable salts thereof, to bedegraded in vivo may be determined employing the Wistar rat model andsubsequent LC/MS analysis (see example 8). Glutaminyl thiazolidine andglutaminyl pyrrolidine were found to be degraded following oraladministration to Wistar rats, to pyroglutaminyl thiazolidine (FIG. 3)and pyroglutaminyl pyrrolidine (FIG. 4), respectively.

A further embodiment of the present invention comprises the use ofcompounds of formula (I), or pharmaceutically acceptable salts thereof,according to any one of the embodiments of the present inventionmentioned above:

in combination with a gene therapeutic expression system for GLP-1comprising a viral vector comprising

-   -   (a) a polynucleotide sequence encoding GLP-1 (gluacogen like        peptide-1); and    -   (b) a polynucleotide sequence encoding a signal sequence        upstream of (a); and    -   (c) a polyadenylation signal downstream of (a); and    -   (d) a polynucleotide sequence encoding a proteolytic cleavage        site located between the polynucleotide sequence encoding GLP-1        and the polynucleotide sequence encoding the signal sequence;        and    -   (e) wherein the expression of GLP-1 underlies a constitutive        promoter or is controlled by a regulatable promotor;    -   (f) wherein, optionally, the viral vector comprises a        polynucleotide sequence encoding GIP (glucose dependent        insulinotropic peptide);    -   (g) wherein, optionally, the viral vector is encompassed by a        mammalian cell.        and/or

in combination with a gene therapeutic expression system for GIPcomprising a viral vector comprising

-   -   (a) a polynucleotide sequence encoding GIP (glucose dependent        insulinotropic peptide); and    -   (b) a polynucleotide sequence encoding a signal sequence        upstream of (a); and    -   (c) a polyadenylation signal downstream of (a); and    -   (d) a polynucleotide sequence encoding a proteolytic cleavage        site located between the polynucleotide sequence encoding GIP        and the polynucleotide sequence encoding the signal sequence;        and    -   (e) wherein the expression of GIP underlies a constitutive        promoter or is controlled by a regulatable promotor;    -   (f) wherein, optionally, the viral vector comprises a        polynucleotide sequence encoding GLP-1 (glucagon like        peptide-1);    -   (g) wherein, optionally, the viral vector is encompassed by a        mammalian cell.

A further embodiment of the present invention comprises the use ofcompounds of formula (I), or pharmaceutically acceptable salts thereof,in combination with a gene therapeutic expression system for GLP-1and/or GIP according to any one of the embodiments of the presentinvention mentioned above wherein:

-   -   the signal sequence upstream of the gene of interest (GLP-1;        GIP) is the murine immunoglobulin κ, signal sequence or the glia        monster exendin signal sequence; and/or    -   the polyadenylation signal downstream of the gene of interest        (GLP-1; GIP) is derived from simian viraus 40 (SV 40); and/or        the proteolytic cleavage site is cleaved by furin preotease;        and/or    -   the gene delivery vector for expression the gene of interest is        an adenoviral, retroviral, leniviral, adeno associated viral        vector; and/or    -   the constitutive promoter is a cytomegalovirus (CMV) promotor,        or a Rous sarcoma long-terminal repeat (LTR) sequence, and the        SV 40 early gene gene promoter; and the inducible promoter is        the Tet-On™/Tet-Off™ system available from Clontech; and/or    -   the mammalian cell is a primate or rodent cell, preferably a        human cell, more preferably a human hepatocyte.

The term “subject” as used herein, refers to an animal, preferably amammal, most preferably a human, who has been the object of treatment,observation or experiment.

The term “therapeutically effective amount” as used herein, means thatamount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue system, animal or human,being sought by a researcher, veterinarian, medical doctor or otherclinician, which includes alleviation of the symptoms of the disease ordisorder being treated.

When used herein the term “conditions associated with diabetes” includesthose conditions associated with the pre-diabetic state, conditionsassociated with diabetes mellitus itself and complications associatedwith diabetes mellitus.

When used herein the term “conditions associated with the pre-diabeticstate” includes conditions such as insulin resistance, includinghereditary insulin resistance, impaired glucose tolerance andhyperinsulinaemia.

“Conditions associated with diabetes mellitus” itself includehyperglycaemia, insulin resistance, including acquired insulinresistance and obesity. Further conditions associated with diabetesmellitus itself include hypertension and cardiovascular disease,especially atherosclerosis and conditions associated with insulinresistance. Conditions associated with insulin resistance includepolycystic ovarian syndrome and steroid induced insulin resistance andgestational diabetes.

“Complications associated with diabetes mellitus” includes renaldisease, especially renal disease associated with Type 2 diabetes,neuropathy and retinopathy.

Renal diseases associated with Type 2 diabetes include nephropathy,glomerulonephritis, glomerular sclerosis, nephrotic syndrome,hypertensive nephrosclerosis and end stage renal disease.

As used herein, the term “pharmaceutically acceptable” embraces bothhuman and veterinary use: for example the term “pharmaceuticallyacceptable” embraces a veterinarily acceptable compound or a compoundacceptable in human medicine a health care.

To prepare the pharmaceutical compositions of this invention, thecompounds of formula (I) or pharmaceutically acceptable salts thereof,optionally in combination with at least on other antidiabetic agent, canbe used as the active ingredient(s). The active ingredient(s) isintimately admixed with a pharmaceutical carrier according toconventional pharmaceutical compounding techniques, which carrier maytake a wide variety of forms depending of the form of preparationdesired for administration, e.g. oral or parenteral such asintramuscular. In preparing the compositions in oral dosage form, any ofthe usual pharmaceutical media may be employed. Thus, for liquid oralpreparations, such as for example, suspensions, elixirs and solutions,suitable carriers and additives include water, glycols, oils, alcohols,flavoring agents, preservatives, coloring agents and the like; for solidoral preparations such as, for example, powders, capsules, gelcaps andtablets, suitable carriers and additives include starches, sugars,diluents, granulating agents, lubricants, binders, disintegrating agentsand the like. Because of their ease in administration, tablets andcapsules represent the most advantageous oral dosage unit form, in whichcase solid pharmaceutical carriers are obviously employed. If desired,tablets may be sugar coated or enteric coated by standard techniques.For parenterals, the carrier will usually comprise sterile water,through other ingredients, for example, for purposes such as aidingsolubility or for preservation, may be included.

Injectable suspensions may also prepared, in which case appropriateliquid carriers, suspending agents and the like may be employed. Thepharmaceutical compositions herein will contain, per dosage unit, e.g.tablet, capsule, powder, injection, teaspoonful and the like, an amountof the active ingredient(s) necessary to deliver an effective dose asdescribed above. The pharmaceutical compositions herein will contain,per dosage unit, e.g., tablet, capsule, powder, injection, suppository,teaspoonful and the like, from about 0.03 mg to 100 mg/kg (preferred0.1-30 mg/kg) and may be given at a dosage of from about 0.1-300 mg/kgper day (preferred 1-50 mg/kg per day) of each active ingredient orcombination thereof. The dosages, however, may be varied depending uponthe requirement of the patients, the severity of the condition beingtreated and the compound being employed. The use of either dailyadministration or post-periotic dosing may be employed.

Preferably these compositions are in unit dosage forms from such astablets, pills, capsules, powders, granules, sterile parenteralsolutions or suspensions, metered aerosol or liquid sprays, drops,ampoules, autoinjector devices or suppositories; for oral parenteral,intranasal, sublingual or rectal administration, or for administrationby inhalation or insufflation. Alternatively, the composition may bepresented in a form suitable for once-weekly or once-monthlyadministration; for example, an insoluble salt of the active compound,such as the decanoate salt, may be adapted to provide a depotpreparation for intramuscular injection. For preparing solidcompositions such as tablets, the principal active ingredient is mixedwith a pharmaceutical carrier, e.g. conventional tableting ingredientssuch as corn starch, lactose, sucrose, sorbitol, talc, stearic acid,magnesium stearate, dicalcium phosphate or gums, and otherpharmaceutical diluents, e.g. water, to form a solid preformulationcomposition containing a homogeneous mixture of a compound of thepresent invention, or a pharmaceutically acceptable salt thereof. Whenreferring to these preformulation compositions as homogeneous, it ismeant that the active ingredient is dispersed evenly throughout thecomposition so that the composition may be readily subdivided intoequally effective dosage forms such as tablets, pills and capsules. Thissolid preformulation composition is then subdivided into unit dosageforms of the type described above containing from 0.1 to about 500 mg ofeach active ingredient or combinations thereof of the present invention.

The tablets or pills of the compositions of the present invention can becoated or otherwise compounded to provide a dosage form affording theadvantage of prolonged action. For example, the tablet or pill cancomprise an inner dosage and an outer dosage component, the latter beingin the form of an envelope over the former. The two components can beseparated by an enteric layer which serves to resist disintegration inthe stomach and permits the inner component to pass intact into theduodenum or to be delayed in release. A variety of material can be usedfor such enteric layers or coatings, such materials including a numberof polymeric acids with such materials as shellac, cetyl alcohol andcellulose acetate.

This liquid forms in which the compositions of the present invention maybe incorporated for administration orally or by injection include,aqueous solutions, suitably flavoured syrups, aqueous or oilsuspensions, and flavoured emulsions with edible oils such as cottonseedoil, sesame oil, coconut oil or peanut oil, as well as elixirs andsimilar pharmaceutical vehicles. Suitable dispersing or suspendingagents for aqueous suspensions, include synthetic and natural gums suchas tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose,methylcellulose, polyvinylpyrrolidone or gelatin.

The method of treating diabetes mellitus, conditions associated withdiabetes mellitus and conditions associated with the pre-diabetic state,as described in the present invention, may also be carried out using apharmaceutical composition comprising a compound of formula (I), or apharmaceutically acceptable salt thereof, optionally in combination withat least one other antidiabetic agent or any other of the compounds asdefined herein and a pharmaceutically acceptable carrier. Thepharmaceutical composition may contain between about 0.01 mg and 100 mg,preferably about 5 to 50 mg, of each compound, and may be constitutedinto any form suitable for the mode of administration selected. Carriersinclude necessary and inert pharmaceutical excipients, including, butnot limited to, binders, suspending agents, lubricants, flavorants,sweeteners, preservatives, dyes, and coatings. Compositions suitable fororal administration include solid forms, such as pills, tablets,caplets, capsules (each including immediate release, timed release andsustained release formulations), granules, and powders, and liquidforms, such as solutions, syrups, elixirs, emulsions, and suspensions.Forms useful for parenteral administration include sterile solutions,emulsions and suspensions.

Advantageously, the compounds of formula (I) and pharmaceuticallyacceptable salts thereof, may be administered in a single daily dose, orthe total daily dosage may be administered in divided doses of two,three or four times daily. Furthermore, the compounds can beadministered in intranasal form via topical use of suitable intranasalvehicles, or via transdermal skin patches well known to those ofordinary skill in that art. To be administered in the form oftransdermal delivery system, the dosage administration will, of course,be continuous rather than intermittent throughout the dosage regimen.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Moreover, when desired or necessary,suitable binders; lubricants, disintegrating agents and coloring agentscan also be incorporated into the mixture. Suitable binders include,without limitation, starch, gelatin, natural sugars such as glucose orbetalactose, corn sweeteners, natural and synthetic gums such as acacia,tragacanth or sodium oleate, sodium stearate, magnesium stearate, sodiumbenzoate, sodium acetate, sodium chloride and the like. Disintegratorsinclude, without limitation, starch, methyl cellulose, agar, bentonite,xanthan gum and the like.

The liquid forms in suitable flavored suspending or dispersing agentssuch as the synthetic and natural gums, for example, tragacanth, acacia,methyl-cellulose and the like. For parenteral administration, sterilesuspensions and solutions are desired. Isotonic preparations whichgenerally contain suitable preservatives are employed when intravenousadministration is desired.

The compounds of formula (I) and the combinations of the presentinvention can also be administered in the form of liposome deliverysystems, such as small unilamellar vesicles, large unilamellar vesicles,and multilamellar vesicles. Liposomes can be formed from a variety ofphospholipids, such as cholesterol, stearylamine orphosphatidylcholines.

The compounds of formula (I) and the combinations of the presentinvention may also be delivered by the use of monoclonal antibodies asindividual carriers to which the compound molecules are coupled. Thecompounds of the present invention may also be coupled with solublepolymers as targetable drug carriers. Such polymers can includepolyvinylpyrrolidone, pyran copolymer,polyhydroxypropylmethacrylamidephenol,polyhydroxyethylaspartamid-ephenol, or polyethyl eneoxidepolyllysinesubstituted with palmitoyl residue. Furthermore, the compounds of thepresent invention may be coupled to a class of biodegradable polymersuseful in achieving controlled release of a drug, for example, polyacticacid, polyepsilon caprolactone, polyhydroxy butyeric acid,polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates andcross-linked or amphipathic block copolymers of hydrogels.

The compounds of formula (I) and the combinations of this invention maybe administered in any of the foregoing compositions and according todosage regimens established in the art whenever treatment of theaddressed disorders is required.

The daily dosage of the products may be varied over a wide range from0.01 to 1,000 mg per mammal per day. For oral administration, thecompositions are preferably provided in the form of tablets containing,0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 150,200, 250 and 500 milligrams of each active ingredient or combinationsthereof for the symptomatic adjustment of the dosage to the patient tobe treated. An effective amount of the drug is ordinarily supplied at adosage level of from about 0.1 mg/kg to about 300 mg/kg of body weightper day. Preferably, the range is from about 1 to about 50 mg/kg of bodyweight per day. The compounds or combinations may be administered on aregimen of 1 to 4 times per day.

Optimal dosages to be administered may be readily determined by thoseskilled in the art, and will vary with the particular compound used, themode of administration, the strength of the preparation, the mode ofadministration, and the advancement of disease condition. In addition,factors associated with the particular patient being treated, includingpatient age, weight, diet and time of administration, will result in theneed to adjust dosages.

The compounds of formula (I), and pharmaceutically acceptable saltsthereof, and the other antidiabetic agent are preferably administeredorally.

Suitably, the particularly beneficial effect on glycaemic controlprovided by the treatment of the invention is an improved therapeuticratio for the combination of the invention relative to the therapeuticratio for one compound of the combination when used alone and at a doseproviding an equivalent efficacy to the combination of the invention.

In a preferred aspect, the particularly beneficial effect on glycaemiccontrol provided by the treatment of the invention may be indicated tobe a synergistic effect relative to the control expected from theeffects of the individual active agents.

In a further aspect of the invention, combining doses of the compoundsof formula (I), or pharmaceutically acceptable salts thereof, and theother antidiabetic agents may produce a greater beneficial effect thancan be achieved for either agent alone at a dose twice that used forthat agent in the combination.

Glycaemic control may be characterised using conventional methods, forexample by measurement of a typically used index of glycaemic controlsuch as fasting plasma glucose or glycosylated haemoglobin (HbA1c). Suchindices are determined using standard methodology, for example thosedescribed in: Tuescher A, Richterich, P., Schweiz. med. Wschr. 101(1971), 345 and 390 and Frank P., ‘Monitoring the Diabetic Patent withGlycosolated Hemoglobin Measurements’, Clinical Products 1988.

The dosage level of each of the active agents when used in accordancewith the methods of the invention may be less than would have beenrequired from a purely additive effect upon glycaemic control.

The methods of the invention may also effect an improvement, relative tothe individual agents, in the levels of advanced glycosylation endproducts (AGEs), and serum lipids including total cholesterol,HDL-cholesterol, LDL-cholesterol including improvements in the ratiosthereof, in particular an improvement in serum lipids including totalcholesterol, HDL-cholesterol, LDL-cholesterol including improvements inthe ratios thereof.

In a further aspect, the invention also provides a process for preparinga pharmaceutical composition comprising a compound of formula (I), or apharmaceutically acceptable salt thereof, another antidiabetic agent anda pharmaceutically acceptable carrier therefor, which process comprisesadmixing the compound of formula (I), or a pharmaceutically acceptablesalt thereof, another antidiabetic agent and a pharmaceuticallyacceptable carrier.

The compositions are preferably in a unit dosage form in an amountappropriate for the relevant daily dosage.

Suitable dosages, including especially unit dosages, of the compounds offormula (I) or the other antidiabetic agent include the known dosagesincluding unit doses for these compounds as described or referred to inreference text such as the British and US Pharmacopoeias, Remington'sPharmaceutical Sciences (Mack Publishing Co.), Martindale The ExtraPharmacopoeia (London, The Pharmaceutical Press) (for example see the31st Edition page 341 and pages cited therein) or the above mentionedpublications.

Thus, suitable dosages for the compounds of formula (I) include thosedisclosed therein, for example 0.01 to 30 mg per day or 0.01 to 10 mgper kilogram of body weight. Also, the suitable doses of the other DP IVinhibitors mentioned herein include those mentioned in the relevantpublications mentioned above.

For the alpha glucosidase inhibitor, a suitable amount of acarbose is inthe range of from 25 to 600 mg, including 50 to 600 mg, for example 100mg or 200 mg.

For the biguanide, a suitable dosage of metformin is between 100 to 3000mg, for example 250, 500 mg, 850 mg or 1000 mg.

For the insulin secretagogue, a suitable amount of glibenclamide is inthe range of from 2.5 to 20 mg, for example 10 mg or 20 mg; a suitableamount of glipizide is in the range of from 2.5 to 40 mg; a suitableamount of gliclazide is in the range of from 40 to 320 mg; a suitableamount of tolazamide is in the range of from 100 to 1000 mg; a suitableamount of tolbutamide is in the range of from 1000 to 3000 mg; asuitable amount of chlorpropamide is in the range of from 100 to 500 mg;and a suitable amount of gliquidone is in the range of from 15 to 180mg. Also a suitable amount of glimepiride is 1 to 6 mg and a suitableamount of glipentide is 2.5 to 20 mg.

A suitable amount of repaglinide is in the range of from 0.5 mg to 20mg, for example 16 mg. Also a suitable amount of nateglinide is 90 to360 mg, for example 270 mg.

In one particular aspect, the composition comprises 2 to 12 mg of5-[4-[2-(N-methyl-N-(2-pyridyl)amino)ethoxy]benzyl]thiazolidine-2,4-dione.

Suitable unit dosages of other insulin sensitisers include from 100 to800 mg of troglitazone such as 200, 400, 600 or 800 mg or from 5 to 50mg, including 10 to 40 mg, of pioglitazone, such as 20, 30 or 40 mg andalso including 15, 30 and 45 mg of pioglitazone.

Suitable dosages of other PPARy agonist insulin sensitisers includethose disclosed for the respective agonist in the abovementionedapplications, for example2-(1-carboxy-2-{4-{2-(5-methyl-2-phenyloxazol-4-yl)ethoxy]phenyl}ethylamino)benzoic acid methyl ester and2(S)-(2-benzoylphenylamino)-3-{4-[2-(5-methyl-2-phenyloxazol-4-yl)ethoxy]phenyl}propionic acid are suitably dosed in accordance withthe dosages disclosed in WO 97/31907.

Also, the dosages of each particular active agent in any givencomposition can as required vary within a range of doses known to berequired in respect of accepted dosage regimens for that compound.Dosages of each active agent can also be adapted as required to takeinto account advantageous effects of combining the agents as mentionedherein.

The compounds of formula (I) or the compositions of the invention may betaken before a meal, while taking a meal or after a meal.

When taken before a meal the compounds of formula (I) or thecompositions of the invention can be taken 1 hour, preferably 30 or even15 or 5 minutes before eating.

When taken whilst eating, the compounds of formula (I) or thecompositions of the invention can be mixed into the meal or taken in aseparate dosage form as described above.

When taken after a meal, the compounds of formula (I) or thecompositions of the invention can be taken 5, 15 or 30 minutes or even 1hour after finishing a meal.

No adverse toxicological effects are expected for the compositions ormethods of the invention in the above mentioned dosage ranges.

All publications, including, but not limited to, patents and patentapplication cited in this specification, are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as fullyset forth.

The invention is illustrated, but not limited by, the followingexamples.

EXAMPLES Example 1 Synthesis of Glutaminyl Pyrrolidine Free Base

N-Benzyloxycarbonylglutamine (2.02 g, 7.21 mmol) was dissolved in 35 mLTHF and cooled to −15° C. CAIBE (isobutylchloroformate) (0.937 mL, 7.21mmol) and 4-methylmorpholine (0.795 mL, 7.21 mmol) were added and thesolution stirred for 15 min. The formation of the mixed anhydride waschecked by TLC (eluent: CHCl₃/MeOH: 9/1). After warming to −10° C.pyrrolidine (0.596 mL, 7.21 mmol) was added. The mixture was brought toroom temperature and stirred overnight. The sediment formed was filteredoff and the solvent was evaporated. The resulting oil was taken up inethylacetate (20 mL) and washed with a saturated solution of sodiumhydrogensulfate followed by a saturated solution of sodium bicarbonate,water and brine. The organic layer was separated, dried and evaporated.The resulting product was checked for purity by TLC (eluent: CHCl₃/MeOH:9/1). Yield: 1.18 g. This product was dissolved in absolute ethanol (40mL). Into the solution ca. 20 mg Pd on charcoal (10%, FLUKA) was addedand the suspension was shaken under a hydrogen atmosphere for 3 h. Theprogress of the reaction was monitored by TLC (eluent: CHCl₃/MeOH: 9/1).After completion of the reaction the catalyst and solvent were removedto give the title compound (99%). The purity was checked by means ofTLC: n-butanol/AcOH/water/ethylacetate: 1/1/1/1, R_(f)=0.4. The identityof the reaction product was checked by NMR analysis.

Example 2 Synthesis of Glutaminyl Thiazolidine Hydrochloride

N-t-Butyloxycarbonylglutamine (2.0 g, 8.12 mmol) was dissolved in TBF (5mL) and cooled to −15° C. CAIBE (isobutylchloroformate) (1.06 mL, 8.12mmol) and 4-methylmorpholine (0.895 mL, 8.12 mmol) were added and thesolution stirred for 15 min, The formation of the mixed anhydride waschecked by TLC (eluent: CHCl₃/MeOH: 9/1). After warming to −10° C.another equivalent of 4-methylmorpholine (0.895 mL, 8.12 mmol) andthiazolidinehydrochloride (1.02 g, 8.12 mmol) was added. The mixture wasbrought to room temperature and stirred overnight. The sediment formedwas filtered off and the solvent was evaporated. The resulting oil wastaken up in chloroform (20 ml) and washed with a saturated solution ofsodium hydrogensulfate followed by a saturated solution of sodiumbicarbonate, water and brine. The organic layer was separated, dried andevaporated. The resulting product was checked for purity by TLC (eluent:CHCl₃/MeOH: 9/1). Yield: 1.64 g. A portion of this product (640 mg) wasdissolved in 3.1 mL ice cold HCl in dioxane (12.98 M, 20 equivalents)and left on ice. The progress of the reaction was monitored by TLC(eluent: CHCl₃/MeOH: 9/1). After completion of the reaction the solventwas removed and the resulting residue was taken up in methanol andevaporated again. The resulting oil was dried over phosphorous-V-oxideand triturated twice with diethylether to give the title compound (0.265g). The purity was checked by HPLC. The identity of the reaction productwas checked by NMR analysis.

Example 3 Synthesis of Glutaminyl Pyrrolidine Hydrochloride

N-t-Butyloxycarbonylglutamine (3.0 g, 12.18 mmol) was dissolved in THF(7 mL) and cooled to −15° C. CAIBE (isobutylchloroformiate) (1.6 mL,12.18 mmol) and 4-methylmorpholine (1.3 mL, 12.18 mmol) were added andthe solution stirred for 15 min. The formation of the mixed anhydridewas checked by TLC (eluent: CHCl₃/MeOH: 9/1). After warming to −10° C. 1equivalent of pyrrolidine (1.0 mL, 12.18 mmol) was added. The mixturewas brought to room temperature and stirred overnight. The sedimentformed was filtered off and the solvent evaporated. The resultingresidue was taken up in chloroform (20 mL) and washed with a saturatedsolution of sodium hydrogensulfate followed by a saturated solution ofsodium bicarbonate, water and brine. The organic layer was separated,dried and evaporated. The resulting product was checked for purity byTLC (eluent: CHCl₃/MeOH: 9/1). The resulting solid (2.7 g) was dissolvedin 13.0 mL ice cold HCl in dioxane (12.98 M, 20 equivalents) and left onice. The progress of the reaction was monitored by TLC (eluent:CHCl₃/MeOH: 9/1). After completion of the reaction the solvent wasremoved and the resulting residue was taken up in methanol andevaporated again. The resulting residue was dried overphosphorous-V-oxide and triturated twice with diethylether to give thetitle compound (980 mg). The purity was checked by HPLC. The identity ofthe reaction product was checked by NMR analysis.

Example 4 K_(i)-Determination

For K_(i)determination of glutaminyl pyrrolidine and glutaminylthiazolidine, dipeptidyl peptidase IV from porcine kidney with aspecific activity against glycylprolyl-4-nitroaniline of 37.5 U/mg andan enzyme concentration of 1.41 mg/mL in the stock solution was used.

100 μL glutaminyl pyrrolidine or glutaminyl thiazolidine in aconcentration range of 1*10⁻⁵ -1*10⁻⁷ M (glutaminyl pyrrolidine) and1*10⁻⁶ M-1*10⁻⁸ M (glutaminyl thiazolidine) respectively were admixedwith 50 μL glycylprolyl-4-nitroaniline in different concentrations (0.4mM, 0.2 mM, 0.1 mM, 0,05 mM) and 100 μl HEPES (40 mM, pH7.6; ionstrength=0.125). The assay mixture was pre-incubated at 30 ° C. for 30min. After pre-incubation, 20 μL DPIV (1:600 diluted) were added andmeasurement of yellow color development due to 4-nitroaniline releasewas performed at 30° C. and λ=405 nm for 10 min using a plate reader(HTS7000 plus, Applied Biosystems, Weiterstadt, Germany). TheK_(i)-values were calculated using Graphit 4.0.15 (Erithacus Software,Ltd, UK) based on a competitive inhibition of DPIV by glutaminylpyrrolidine or glutaminyl thiazolidine. They were determined forglutaminyl thiazolidine as K_(i)=3.12*10⁻⁷ M±5.11*10⁻¹⁰ M and forglutaminyl pyrrolidine as K_(i)=1.30*10⁻⁶ M±8.49*10⁻⁸ M.

Example 5 K_(i)-Determination in Human Plasma

Human plasma contains N-terminal Xaa-Pro releasing activity. 70 μLglutaminyl pyrrolidine or glutaminyl thiazolidine in an concentrationrange of 1*10⁻⁵ M -1*10⁻⁷ M (glutaminyl pyrrolidine) and 1*10⁻⁶ M-1*10⁻⁸M (glutaminyl thiazolidine) respectively were admixed with 50 μLglycylprolyl-4-nitroaniline in different concentrations (0.4 mM, 0.2 mM,0.1 mM, 0,05 mM) and 100 μl HEPES (40 mM, pH7.6). The assay mixture waspre-incubated at 30 ° C. for 5 min and 22 hours respectively. Afterpre-incubation, 50 μL human plasma were added and measurement of yellowcolor development due to 4-nitroaniline release was performed at 30° C.and λ=405 nm for 10 min using a plate reader ([HTS7000 plus, AppliedBiosystems, Weiterstadt, Germany). The K_(i)-values were calculatedusing Graphit 4.0.15 (Erithacus Software, Ltd, UK) based on acompetitive inhibition of DPIV by glutaminyl pyrrolidine or glutaminylthiazolidine. They were determined for glutaminyl thiazolidine asK_(i)=4.03*10⁻⁷ M±2.19*10⁻¹⁰ M after 5 min 5.13*10⁻⁷ M±1.26*10⁻⁸ M after22 hours pre-incubation and for glutaminyl pyrrolidine asK_(i)=1.30*10⁻⁶ M±4.89*10⁻⁸ M after 5 min and 1.36*10⁻⁶ M±3.21*10⁻⁸ Mafter 22 hours pre-incubation.

Example 6 Dose Escalation Study in Fatty Zucker Rats After OralAdministration of Glutaminyl Pyrrolidine

N=30 male Zucker rats (fa/fa), mean age 11 weeks (5-12 weeks), mean bodyweight 350 g (150-400 g), were purchased from Charles River (Sulzfeld,Germany). After delivery they were kept for >12 weeks until nearly allfatty Zucker rats had the characteristics of manifest diabetes mellitus.A group of N=8 animals were recruited for testing three escalating dosesof glutaminyl pyrrolidine vs. placebo (saline). Animals weresingle-caged under standardized conditions with controlled temperature(22±2° C.) on a 12/12 hours light/dark cycle (light on at 06:00 AM).Sterile standard pelleted chow (ssniff® Soest, Germany) and tap wateracidified with HCl were allowed ad libitum. Fatty Zucker rats of 24-31weeks (mean: 25 weeks) age, adapted to the housing conditions, were wellprepared for the study. Catheters were implanted into the carotid arteryof fatty Zucker rats under general anaesthesia (i.p. injection of 0.25ml/kg b.w. Rompun®[2%], BayerVital, Germany and 0.5 ml/kg b.w. Ketamin10, Atarost GmbH & Co., Twistringen, Germany). The animals were allowedto recover for one week. The catheters were flushed with heparin-saline(100 IU/ml) three times per week.

Placebo (1 ml saline, 0.154 mol/l) or escalating doses of glutaminylpyrrolidine (5, 15 and 50 mg/kg b.w.) were administered to groups of N=8fatty Zucker rats. 375 mg of glutaminyl pyrrolidine were dissolved in1000 μl DMSO (E. Merck, Darmstadt; Germany [Dimethyl sulfoxide p.a.]).10 mL saline was added and 1 ml aliquots, each containing 34.09 mg ofglutaminyl pyrrolidine, were stored at −20° C. For preparation of thetest substance, dose dependent aliquots were diluted in saline. Afterovernight fasting, placebo or test substance were administered to thefatty Zucker rats via feeding tube orally (15 G, 75 mm; Fine ScienceTools, Heidelberg, Germany) at −10 min An oral glucose tolerance test(OGTT) with 2 g/kg b.w. glucose (40% solution, B. Braun Melsungen,Melsungen, Germany) was administered at ±O min via a second feedingtube. Venous blood samples from the tail veins were collected at −30min, −15 min, ±0 min and at 5, 10, 15, 20, 30, 40, 60, 90 and 120 mininto 20 lμl glass capillaries, which were placed in standard tubesfilled with 1 mL solution for blood glucose measurement. All bloodsamples were labelled with Code number, Animal Number, Date of samplingand Time of sampling.

Glucose levels were measured using the glucose oxidase procedure (SuperG Glucose analyzer; Dr. Müller Gerältebau, Freital, Germany).

Statistical evaluations and graphics were performed with PRISM® 3.02(GraphPad Software, Inc.). All parameters were analysed in a descriptivemanner including mean and SD.

The placebo treated diabetic Zucker rats showed a strongly elevatedblood glucose excursion indicating glucose intolerance of manifestdiabetes mellitus. Administration of 5 mg/kg b.w. glutaminyl pyrrolidineresulted in a limited improvement of glucose tolerance in diabeticZucker rats. Significant lowering of elevated blood glucose levels andimprovement of glucose tolerance was achieved after administration of 15mg/kg and 50 mg/kg b.w. glutaminyl pyrrolidine (see FIG. 3).

Example 7 Dose Escalation Study in Fatty Zucker Rats After OralAdministration of Glutaminyl Thiazolidine

N=30 male Zucker rats (fa/fa), mean age 11 weeks (5-12 weeks), mean bodyweight 350 g (150-400 g), were purchased from Charles River (Sulzfeld,Germany). After delivery they were kept for >12 weeks until nearly allfatty Zucker rats had the characteristics of manifest diabetes mellitus.A group of N=8 animals were recruited for testing three escalating dosesof glutaminyl thiazolidine vs. placebo (saline). Animals weresingle-caged under standardized conditions with controlled temperature(22±2° C.) on a 12/12 hours light/dark cycle (light on at 06:00 AM).Sterile standard pelleted chow (ssniff® Soest, Germany) and tap wateracidified with HCl were allowed ad libitum. Fatty Zucker rats of 24-31weeks (mean: 25 weeks) age, adapted to the housing conditions, were wellprepared for the study. Catheters were implanted into the carotid arteryof fatty Zucker rats under general anaesthesia (i.p. injection of 0.25ml/kg b.w. Rompun® [2%], BayerVital, Germany and 0.5 ml/kg b.w. Ketamin10, Atarost GmbH & Co., Twistringen, Germany). The animals were allowedto recover for one week. The catheters were flushed with heparin-saline(100 IU/ml) three times per week.

Placebo (1 mL saline, 0.154 mol/L) or escalating doses of glutaminylthiazolidine (5, 15 and 50 mg/kg b.w.) were administered to groups ofN=8 fatty Zucker rats. The respective amounts of glutaminyl thiazolidinewere dissolved in 1000 μl saline. After overnight fasting, placebo ortest substance was administered to the fatty Zucker rats via feedingtube orally (15 G, 75 mm; Fine Science Tools, Heidelberg, Germany) at−10 min An oral glucose tolerance test (OGTT) with 2 g/kg b.w. glucose(40% solution, B. Braun Melsungen, Melsungen, Germany) was administeredat ±0 min via a second feeding tube. Venous blood samples from the tailveins were collected at −30 min, −15 min, ±0 min and at 5, 10, 15, 20,30, 40, 60, 90 and 120 min into 20 μL glass capillaries, which wereplaced in standard tubes filled with 1 ml solution for blood glucosemeasurement. All blood samples were labelled with Code number, AnimalNumber, Date of sampling and Time of sampling.

Glucose levels were measured using the glucose oxidase procedure (SuperG Glucose analyzer; Dr. Müiller Gerätebau, Freital, Germany).

Statistical evaluations and graphics were performed with PRISM® 3.02(GraphPad Software, Inc.). All parameters were analysed in a descriptivemanner including mean and SD.

The placebo treated diabetic Zucker rats showed a strongly elevatedblood glucose excursion indicating glucose intolerance of manifestdiabetes mellitus. Administration of 5 mg/kg b.w., 15 mg/kg and 50 mg/kgb.w glutaminyl thiazolidine resulted in a dose dependent lowering ofelevated blood glucose levels and improvement of glucose tolerance indiabetic Zucker rats (see FIG. 4).

Example 8 In Vivo Inactivation of Glutaminyl Thiazolidine After OralAdministration to Wistar Rats

Glutaminyl thiazolidine was administered to Wistar rats orally. Afterapplication of placebo or glutaminyl thiazolidine, arterial bloodsamples were taken at 2.5, 5, 7.5, 10, 15, 20, 40, 60 and 120 min fromthe carotid catheter of the conscious unrestrained rats to determine theformation of degradation products of glutaminyl thiazolidine. Foranalysis, simple solid phase extraction procedure on C18 cartridges wasused to isolate the compounds of interest from the plasma. The extractswere analysed using reversed-phase liquid chromatography on Lichrospher60 RP Select B column hyphenated with tandem mass spectrometry operatingin the APCI positive mode. An internal standard method was used forquantification.

After oral administration of glutaminyl thiazolidine to Wistar rats, adegradation of the compound was found. Using LC/MS, the degradationproduct could be defined as pyroglutaminyl thiazolidine. See FIGS. 3 and5.

Example 9 Determination of DPIV Inhibiting Activity of GlutaminylPyrrolidine and Glutaminyl Thiazolidine After Intravasal and OralAdministration to Wistar Rats

Male Wistar rats (Shoe: Wist(Sho)) with a body weight ranging between250 and 350 g were purchased from Tierzucht Schönwalde (Schönwalde,Germany). Animals were single-caged under conventional conditions withcontrolled temperature (22±2° C.) on a 12/12 hours light/dark cycle(light on at 06:00 AM). Standard pelleted chow (ssniff® Soest, Germany)and tap water acidified with HCl were allowed ad libitum. After ≧oneweek of adaptation at the housing conditions, catheters were implantedinto the carotid artery of Wistar rats under general anaesthesia (i.p.injection of 0.25 ml/kg b.w. Rompun®[2%], BayerVital, Germany and 0.5ml/kg b.w. Ketamin 10, Atarost GmbH & Co., Twistringen, Germany). Theanimals were allowed to recover for one week. The catheters were flushedwith heparin-saline (100 IU/ml) three times per week. In case ofcatheter dysfunction, a second catheter was inserted into thecontra-lateral carotid artery of the respective rat. After one week ofrecovery from surgery, this animal was reintegrated into the study. Incase of dysfunction of the second catheter, the animal was withdrawnfrom the study. A new animal was recruited and the experiments werecontinued in the planned sequence, beginning at least 7 days aftercatheter implantation.

To rats with intact catheter finction were administered placebo (1 mLsaline, 0.154 mol/l) or 100 mg/kg b.w. glutaminyl pyrrolidine or 100mg/kg b.w. glutaminyl thiazolidine via the oral and the intra-vasal(intra-arterial) route. After overnight fasting, 100 μL samples ofheparinised arterial blood were collected at −30, −5, and 0 min. Thetest substance was dissolved freshly in 1.0 mL saline (0.154 mol/l) andwas administered at 0 min either orally via a feeding tube (75 mm; FineScience Tools, Heidelberg, Germany) or via the intra-vasal route. In thecase of oral administration, an additional volume of 1 mL saline wasinjected into the arterial catheter. In the case of intra-arterialadministration, the catheter was immediately flushed with 30 μL salineand an additional 1 mL of saline was given orally via the feeding tube.After application of placebo or the test substances, arterial bloodsamples were taken at 2.5, 5, 7.5, 10, 15, 20, 40, 60 and 120 min fromthe carotid catheter of the conscious unrestrained rats. All bloodsamples were collected into ice cooled Eppendorf tubes(Eppendorf-Netheler-Hinz, Hamburg, Germany) filled with 10 μL 1M sodiumcitrate buffer (pH 3.0) for plasma DPIV activity measurement. Eppendorftubes were centrifuged immediately (12000 rpm for 2 min, HettichZentrifuge EBA 12, Tuttlingen; Germany): The plasma fractions werestored on ice until analysis or were frozen at −20° C. until analysis.All plasma samples were labelled with Code number, Animal Number, Dateof sampling and Time of sampling.

The assay mixture for determination of plasma DPIV activity consisted of80 μL reagent and 20 μL plasma sample. Kinetic measurement of theformation of the yellow product 4-nitroaniline from the substrateglycylprolyl-4-nitroaniline was performed at 390 nm for 1 min at 30° C.after 2 min pre-incubation at the same temperature. The DPIV activitywas expressed in mU/mL.

Statistical evaluations and graphics were performed with PRISM® 3.02(GraphPad Software, Inc.). All parameters were analysed in a descriptivemanner including mean and SD.

The compounds glutaminyl pyrrolidine and glutaminyl thiazolidine in adose of 100 mg/kg b.w. vs. placebo inhibited plasma DPIV activity afteroral and intra-vasal administration.

Example 10 Effect of Glutaminyl Thiazoildine Hydrochloride and MetforminEither Alone or in Combination on Glycaemic Control in Diet-InducedObese Rats With Impaired Glucose Tolerance

Selectively bred male rats, 5-6 weeks of age, displaying enhancedlikelihood of developing diet-induced obesity (DIO) are selected fromthe breeding colony. A total of 40 DIO animals are included in thestudy. The DIO rats are chosen because they are likely to reflect thesegment of the human population, who develop obesity and later typediabetes upon exposure to high-calorie fat rich diet.

Upon entry to the experiment, rats are housed individually (1 rat/cage)in a 12/12 light-dark cyldle (light from 0600-1800 h) with controlledtemperature conditions (22-24° C.). At this time rats are offered Highfat (HF) diet (4.41 kcal/g−Energy %: Carbohydrate 51.4 kcal %, Fat 31.8kcal %, Protein 16.8 kcal %; diet #12266B; Research Diets, New Jersey,USA; the HF diet ensure sufficient intake of vitamins and traceelements) and water ad libitum. After one week of acclimatisation, 24 hfood and water intake and body-weight is measured gravimetrically twiceweekly (in the morning between 8-10 am). After 3 weeks of HF feeding,average daily food consumption is calculated for all rats. The averagefood intake comprises a platform from which a scheduled feeding regimeis implemented. Animals are offered 75% of the daily average foodconsumption from 8:00-12:00 AM, and 25% of the daily average foodconsumption from 4:00 PM-8:00 PM:

After 3 weeks of schedule feeding, animals are stratified according toweight. At day 0, animals are randomised (n=10 in each group) toparticipate in one of following drug treatment groups: Group A: vehicle(distilled water) Group B: glutaminyl thiazolidine hydrochloride (60mg/kg BID) Group C: Metformin (125 mg/kg BID) Group D: glutaminylthiazolidine hydrochloride (60 mg/kg BID) + Metformin (125 mg/kg BID)

All drugs are given orally by gavage, volume 200 μl, twice daily (8:00AM and 4:00 PM). This mode of administration ensures that all animalsreceive the same amount of drug irrespective of the diet eaten therebyensuring more accurate comparison between the chow and high fat diet fedgroups. Animals receive two daily doses of either compound for a totalof 42 days (day 1-42). On days 6 and 40 animals are subjected to an oralglucose tolerance test (OGTT). Two days later, on day 42, treatment isdiscontinued and animals are followed drug free for yet another day(still following the schedule feeding regime). On day 43, animals aresacrificed in a semi-starved state as they have had access to only 25%of their daily energy requirement from 12: AM the previous day. In themorning period (from 8-12 AM), animals are anaesthetised by CO₂inhalation and blood samples are collected. Optionally, tissue samplescan be taken and rapidly frozen in liquid nitrogen for later analysis oftissue specific gene expression and lipid content. Blood and tissuesampling will be carried out in a room adjacent to the permanent stablein order to ensure lowest possible level of stress. Fat samples areweighed and frozen such that accurate analysis of fat depots can becarried out. Fat depot analysis could be carried out by removingmesenterial, retroperitoneal, epididymal and subcutaneous inguinal fat.

Analytical Methods:

Oral Glucose Tolerance Test (OGTT):

This test is carried out at 8:00 AM on days 6 and 42. Animals are mildlyfasted as they have had access to only 25% of their daily energyrequirements in the preceding 20 hrs (Since 12:00 AM the previous day).Blood samples are taken from an indwelling arterial catheter andP-glucose is measured on automated analyser (Roche Diagnostics) at timepoints −60, −30, 0, 15, 30, 60, 120, and 180 min after oraladministration of 1 g/kg glucose (using 1 g/ml dH₂O). The oral glucoseload is given as gavage via a duodenally placed tube connected to asyringe ensuring accurate dosing. P-insulin is measured at time points:0, 15, 30, 60, 120 using an ultra-sensitive ELISA (Shibayagi,Japan).

Blood Sampling and Plasma Measurements:

All rats are equipped with intra-arterial catheters at day −7. Theintra-arterial catheters are positioned in the abdominal aorta via thefemoral artery and kept patent by injection of heparinised saline at theend of all sampling procedures. All blood samples are taken in EDTAVacutainer tubes and plasma glucose is measured together with totalCholesterol and triacylglycerol. Optionally, as a reflection oflipolysis, we could measure plasma levels of glycerol. On the day ofsacrifice, heart puncture blood is collected in three tubes:Vacutainer-EDTA; Vacutainer-EDTA+1% NaF; Vacutainer-EDTA+Aprotinin (750KIU).

Various blood sample “packages” are taken:

-   A) Glycaemic profile: fasting P-glucose, P-insulin and HbA1c-   B) 24 hour glycaemic profile: B-glucose every 3^(rd)hour (8:00,    11:00, 14:00, 17:00, 20:00, 23:00, 02:00, 05:00). Alternatively,    P-glucose and P-insulin with same time profile-   C) Meal associated glucose: B-glucose before and after morning meal    (8:00 AM and 12:00 AM)-   D) Fasting glucose & lipids: Fasting-P-glucose, P-triacylglycerol,    P-total cholesterol-   E) OGTT: for details see above

Plasma-Glucose, HbA1c, Plasma-total Cholesterol, Plasma-triacylglycerolis measured using standard enzyme assay kits on a fully automatedanalyser (Roche Diagnostics). Plasma non-esterified free fatty acids(NEFA) are determined by a spectrophotometer using acyl-CoA oxidasebased colorimetric kit (NEFA-C, WAKO pure chemicals, Osaka, Japan).Samples taken in Vacuatiner-EDTA+1% NaF are used for FFA analyses.

Plasma insulin is measured with an ultra-sensitive ELISA based assay(Shibayagi, Japan). Bioactive GLP-1(7-37) and total GLP-1immunoreactivities are measured with a Linco multiple ELISA kit (LincoResearch Immunoassay, St. Charles, Mo.).

Data, Reporting, and Statistical Evaluation:

All data is fed into Excel 97 or 2000 spread sheets and subsequentlysubjected to relevant statistical analyses (Statview or Graph Padsoftware). Results are presented as mean±SEM (standard error of themean) unless otherwise stated. Statistical evaluation of the data iscarried out using one-way analysis of variance (ANOVA) with appropriatepost-hoc analysis between control and treatment groups in cases wherestatistical significance is established (p<0.05).

Results:

Using a protocol of this type both glutaminyl thiazolidine andglutaminyl thiazolidine in combination with Metformin resulted inimproved oral glucose tolerance.

Example 11 Inhibition of DP IV-Like Enzymes—Dipeptidyl Peptidase II

DP II (3.4.14.2) releases N-terminal dipeptides from oligopeptides ifthe N-terminus is not protonated (McDonald, J. K., Ellis, S. & Reilly,T. J., 1966, J Biol. Chem., 241, 1494-1501). Pro and Ala in P₁-positionare preferred residues. The enzyme activity is described as DPIV-likeactivity, but DP II has an acidic pH-optimum. The enzyme used waspurified from porcine kidney. 100 μL glutaminyl pyrrolidine orglutaminyl thiazolidine in an concentration range of 1*10⁻⁴M-5*10⁻⁸Mwere admixed with 100 μL buffer solution (40 mM HEPES, pH 7.6, 0.015%Brij, 1 mM DTT), 50 μL lysylalanylaminomethylcoumarine solution (5 mM)and 20 μl porcine DP II (250 fold diluted in buffer solution).Fluorescence measurement was performed at 30° C. and λ_(exiatation)=380nm, λ_(emission)=465 nm for 25 min using a plate reader (HTS7000 plus,Applied Biosystems, Weiterstadt, Germany). The K_(i)-values werecalculated using Graphit 4.0.15 (Erithacus Software, Ltd., UK) and weredetermined as K_(i=)8.52*10⁻⁵ M±6.33*10⁻⁶ M for glutaminyl pyrrolidineand K_(i=)1.07*10⁻⁵ M±3.81*10⁻⁷ M for glutaminyl thiazolidine.

Example 12 Cross Reacting Enzymes

Glutaminyl pyrrolidine or glutaminyl thiazolidine were tested for theircross reacting potency against dipeptidyl peptidase I, prolyloligopeptidase and prolidase.

Dipeptidyl Peptidase I (DP L Cathepsin C)

DP I or cathepsin C is a lysosomal cysteine protease which cleavesdipeptides from the N-terminus of their substrates (Gutman, H. R. &Fruton, J. S., 1948, J Biol. Chem., 174, 851-858). It is classified as acysteine protease. The enzyme used was purchased from Qiagen (QiagenGmbH, Hilden, Germany). In order to get a fully active enzyme, theenzyme was diluted 1000 fold in MES buffer pH5.6 (40 mM MES, 4 mM DTT, 4mM KCl, 2 mM EDTA, 0.015% Brij) and pre-incubated for 30 min at 30° C.50 μL glutaminyl pyrrolidine or glutaminyl thiazolidine in aconcentration range of 1*10⁻⁵ M-1*10⁻⁷ M were admixed with 110 μLbuffer-enzyme-mixture. The assay mixture was pre-incubated at 30° C. for15 min After pre-incubation, 100 μL histidylseryl-para-nitroanilide(2*10⁻⁵ M) were added and measurement of yellow color development due topara-nitroaniline release was performed at 30° C. and λ_(excitation)=380nm, λ_(emission)=465 nm for 10 min, using a plate reader (HTS7000 plus,Applied Biosystems, Weiterstadt, Germany). The IC₅₀-values werecalculated using Graphit 4.0.15 (Erithacus Software, Ltd., UK). Noinhibition of the DP I enzyme activity by glutaminyl pyrrolidine orglutaminyl thiazolidine was found.

Prolyl Oligopeptidase (POP)

Prolyl oligopeptidase (EC 3.4.21.26) is a serine type endoprotease whichcleaves off peptides at the N-terminal part of the Xaa-Pro bond (Walter,R., Shlank, H., Glass, J. D., Schwartz, I. L. & Kerenyi, T. D., 1971,Science, 173, 827-829). Substrates are peptides with a molecular weightup to 3000 Da. The enzyme used was a recombinant human prolyloligopeptidase. Recombinant expression was performed in E. coli understandard conditions as described elsewhere in the state of the art. 100μL glutaminyl pyrrolidine or glutaminyl thiazolidine in an concentrationrange of 1*10⁻⁴ M-5*10⁻⁸ M were admixed with 100 μL buffer solution (40mM HEPES, pH 7.6, 0.015% Brij, 1 mM DTT) and 20 μL POP solution. Theassay mixture was pre-incubated at 30° C. for 15 min Afterpre-incubation, 50 μL glycylprolylprolyl-4-nitroaniline solution (0.29mM) were added and measurement of yellow color development due to4-nitroaniline release was performed at 30° C. and λ=405 nm for 10 minusing a plate reader (sunrise, Tecan, Crailsheim, Germany). The IC₅₀-values were calculated using Graphit 4.0.15 (Erithacus Software,Ltd., UK). No inhibition of POP activity by glutaminyl pyrrolidine orglutaminyl thiazolidine was found.

Prolidase (X-Pro Dipeptidase)

Prolidase (EC 3.4.13.9) was first described by Bergmann & Fruton(Bergmann, M. & Fruton, J. S., 1937, J Biol. Chem. 189-202). Prolidasereleases the N-terminal amino acid from Xaa-Pro dipeptides and has a pHoptimum between 6 and 9. Prolidase from porcine kidney (ICN Biomedicals,Eschwege, Germany). was solved (1 mg/mL) in assay buffer (20 mMNH₄(CH₃COO)₂, 3 mM MnCl₂, pH 7.6). In order to get a fully active enzymethe solution was incubated for 60 min at room temperature. 450 μLglutaminyl pyrrolidine or glutaminyl thiazolidine in an concentrationrange of 5*10⁻³ M-5*10⁻⁷ M were admixed with 500 μL buffer solution (20mM NH₄(CH₃COO)₂, pH 7.6) and 250 μL Ile-Pro-OH (0.5 mM in the assaymixture). The assay mixture was pre-incubated at 30° C. for 5 min Afterpre-incubation, 75 μL Prolidase (1:10 diluted in assay buffer) wereadded and measurement was performed at 30° C. and λ=220 nm for 20 minusing a UV/Vis photometer, UVW (Thermo Spectronic, Cambridge, UK). TheIC₅₀-values were calculated using Graphit 4.0.15 (Erithacus Software,Ltd., UK). They were determined as IC 50>3 mM for glutaminylthiazolidine and as IC₅₀=3.4*10⁻⁴ M±5.63*10⁻⁵ for glutaminylpyrrolidine.

Example 13 Plasma Stability

In order to investigate the stability of glutaminyl pyrrolidine orglutaminyl thiazolidine in human plasma, the activity of DPIV in plasmawas determined at a defined time. The average DPIV activity in humanplasma was determined as 43.69 U/mL. In the working solution, the plasmawas diluted in 0.9% NaCl to fix the DPIV activity level at 25 U/mL.Plasma and glutaminyl pyrrolidine or glutaminyl thiazolidine indifferent concentrations (5*10⁻⁵, 2.5*10⁻⁵, 1.25*10⁻⁵ M in plasma) wereincubated at 37° C. At defined time points samples were taken using apipette roboter (Gilson 215, Liquid handler, Gilson) and transferred ina microtiter plate containing 5*10⁻⁵ M glycylprolylaminomethylcoumarinein 0.9% NaCl+015% Brij per well. After 6 min the reaction was stopped byaddition of isoleucylthiazolidine (5*10⁻⁵ M in 0.9% NaCl solution).Fluorescence measurement was performed against 0.9% NaCl in plasma(reference standard) using a plate reader (HTS7000 plus, AppliedBiosystems, Weiterstadt, Germany). The half-life of the inhibitorypotency of glutaminyl pyrrolidine or glutaminyl thiazolidine wascalculated by plotting the enzyme activity versus reaction time. Forboth compounds, no half-time could be determined. The substance isconsidered to be stable in human plasma over 22 hours.

Example 14 Synthesis of Other Salt Forms of Glutaminyl Thiazolidine

Glutaminyl thiazolidine hydrochloride (1 g, 3.43 mmol) was applied on astrong basic ion exchange column (DOWEX® 550 A, 10 mL dry material,preconditioned as described). The fractions were collected and titratedwith 1N HCl against bromthymolblue in order to estimate the content offree base. After that the corresponding amount of the required acid wasadded and the solution was lyophilized. The resulting material wasre-crystallized from methanol/ether.

The 3,5-di-tertbutylbenzoate and sulfinate salts of glutaminylthiazolidine are novel and as such form a fiuther aspect of the presentinvention.

Characterization of different acid addition salts of glutaminylthiazdlidine: Salt PGT MP (° C.) Appearance Hydrochloride <0.5 167-169crystalline Fumarate 0.2 128-131 crystalline Benzoate 0.6 116-118crystalline Maleinate 4.6 128-132 crystalline Oxalate 0 Broad, amorphous 90-110 3,5-Di-tert-butylbenzoate 1.06 Sharp, crystalline 125 SulfinateUnknown by- 143-145 crystalline product Salicylate 0.6   120-127.6crystalline Acetate 0.1 88-89 amorphousPGT pyroglutaminyl thiazolidine, area % determined by HPLC analysisMP melting point

Example 15 Effects of Glutaminyl Thiazolidine and Metformin Either Aloneor in Combination on Glycemic Control in Diabetic Zucker (fa/fa) Rats

Ten or eleven weeks old male Zucker (fa/fa) rats were purchased fromCharles River (Sulzfeld, Germany). Animals were kept under standardizedsemi-barrier conditions with controlled temperature (22±2° C.) on a12/12 hours light/dark cycle (light on at 06:00 a.m.). Standard pelletedchow (ssniff®, Soest, Germany) and tap water acidified with HCl wereallowed ad libitum. At the age of 12 weeks the animals (N=42) weredivided in random order into six experimental groups to be medicated.Definition of the Experimental Groups for the Medications (GT=glutaminylthiazolidine): Group CO (N = 7): placebo (distilled water), b.i.d., oralGroup GT (N = 7): 60 mg/kg b.w. GT, b.i.d., oral Group Met-low (N = 7):125 mg/kg b.w. Metformin, b.i.d., oral Group Met-high (N = 7): 300 mg/kgb.w. Metformin, b.i.d., oral Group GT + Met-low (N = 7): 60 mg/kg b.w.GT + 125 mg Metformin, b.i.d., oral Group GT + Met-high (N = 7) 60 mg/kgb.w. GT + 300 mg Metformin, b.i.d., oral

The single or combined doses per kg b.w. were solved in 5 mL dist. waterfor oral administration.

Experimental procedures:

First OGTT:

The study was started with the 12 to 13 weeks old Zucker (fa/fa) rats.At the beginning of the study an OGTT was performed (2 g glucose/kg bodyweight (b.w.); administration volume: 5 mL/kg of a 40% solution; B.Braun Melsungen, Melsungen, Germany) after a 16 h fast. The glucose wasadministered via a feeding tube (15 g, 75 mm; Fine Science Tools,Heidelberg, Germany). Dosing was performed at t=5 min before OGTT bygavage.

Group characterization and medication for the first and second OGTTAnimals Group (N) Drug/Dose Comments CO 7 Placebo; 5 ml/kg b.w. 0.5mL/100 g b.w. GT 7 P93/01; 60 mg/kg b.w. 60 mg P93/01 solved in 5 mLdist. water, 0.5 mL/100 g b.w Met-low 7 Metformin; 125 mg/kg b.w. 125 mgMetformin solved in 5 mL dist. water, 0.5 mL/100 g b.w Met-high 7Metformin; 300 mg/kg b.w. 300 mg Metformin solved in 5 mL dist. water,0.5 mL/100 g b.w GT + Met-low 7 GT; 60 mg/kg b.w. + Metformin; 60 mgGT + 125 mg Metformin 125 mg/kg b.w. solved in 5 mL dist. water, 0.5mL/100 g b.w GT + Met-high 7 GT; 60 mg/kg b.w. + Metformin; 60 mg GT +300 mg Metformin 300 mg/kg b.w. solved in 5 mL dist. water, 0.5 mL/100 gb.w

Blood samples were taken from tail veins to measure blood glucose andserum insulin at −15, ±0 min, 15, 30, 60, 90, 120 and 180 min (thelatter time without insulin samples) with respect to time of glucoseadministration.

After the first OGTT the animals in the groups were dosed twice dailywith the respective drugs at 08:00 AM and at 04:00 PM, respectively:

During the two weeks of medication morning blood glucose was measuredbefore the 08:00 AM medication at Monday, Wednesday and Friday.

Food intake was determined every day during the time of medication.

All the animals were weighted three times per week at 7:30 AM.

A second OGTT was performed after two weeks of medication (Day 15). Thefood was withdrawn at 04:00 PM the day before (16 h fast). The OGTT wasperformed with pre-medication at −5 min and oral glucose loading at ±0min. Blood samples were taken from tail veins to measure blood glucose,and serum insulin_(e) at −15, ±0 min, 15, 30, 60, 90, 120 and 180 min(the latter time without insulin samples).

Glycated hemoglobin was measured before (Day −7) and on Day 18.

Measurements:

Glucose-For determination of glucose 20 μL blood were collected at −15,±0 min (before OGTT) and 15, 30, 60, 90, 120 and 180 min post OGTT.

Insulin-Insulin concentrations were assayed by the antibody RIA method(Linco Research, Inc. St. Charles, Mo., USA).

Glycated haemoglobin-Percentage of glycated hemoglobin A (HbA1c) wasestimated with the “DCA 2000R Hämoglobin A1c-Reagenz kit”(Bayer VitalGmbH, Fernwald, Germany).

The body weight was measured using a platform balance (Scaltec,Heiligenstadt, Germany).

Mixed venous blood samples from the tails were collected into 20 μLglass capillaries, which were placed in standard tubes filled with 1 mlsolution for hemolysis (blood glucose measurement) and in sample tubesfor serum insulin (50 μL blood).

Raw data of glucose analysis were provided by IDK to probiodrug in Excelformat as soon as possible. Data for each drug and each parameter(glucose, insulin) were summarized by descriptive statistics (mean,SEM). AUC and baseline corrected AUC (baseline was set to the value att=0 min) were calculated. Changes from baseline were calculated andsummarized by descriptive statistic.

Results:

Subchronic (18 days) b.i.d. administration of glutaminyl thiazolidinealone or in combination with Metformin to diabetic fatty Zucker rats(fa/fa) resulted in an improved glucose tolerance. Drug administrationhad no affected food and water intake in all experimental groups. GT andMet-low groups showed significantly improved glucose tolerance curves inthe OGTT and the reactive and absolute G-AUC were significantly reduced(p<0.05 vs. Control). Met-high, GT+Met-low and GT+Met-high groupsrevealed a further improvement of glucose tolerance curve and thereactive and absolute G-AUC were once more lowered (p<0.05 vs. Control).FIG. 6 shows baseline corrected glucose AUC during first OGTT in fastedZucker rats loaded with placebo, GT, Met-low, Met-high, GT+Met-low andGT+Met-high (at −5 min) and OGTT (at 0 min) after 14 days of medication(baseline was set as y-value at t=O min).

Example 16 Effects of Combination Therapy of Glutaminyl Thiazolidinewith Other Oral Antidiabetics

Male, eight weeks old male Zucker (fa/fa) rats were kept understandardized semi-barrier conditions with controlled temperature (22±2°C.) on a 12/12 hours light/dark cycle (light on at 06:00 a.m.). Standardpelleted chow (ssniff®, Soest, Germany) and tap water acidified with HClwere allowed ad libitum. At the age of 12 weeks the animals (N=42) weredivided in random order into six experimental groups to be medicated.The experimental groups for the two weeks of medication are as follows(GT=glutaminyl thiazolidine): Group CO (N = 5): placebo (distilledwater), b.i.d., oral at 08.00 AM and 04.00 PM Group GT (N = 5) 60 mg/kgb.w. GT b.i.d., oral at 08.00 AM and 04.00 PM. Group Rosiglitazone + GT(N = 5): 3 mg/kg b.w. Rosiglitazone, once per day p.o. at 08.00 AM + 60mg/kg b.w. GT, b.i.d., oral at 08.00 AM and 04.00 PM. Group Acarbose +GT (N = 5): 40 mg acarbose/100 g chow with free (food) access + 60 mg/kgb.w. GT, b.i.d., oral at 08.00 AM and 04.00 PM. Group Glibenclamide + GT(N = 5): 5 mg/kg b.w. glibenclamide, b.i.d., oral + 60 mg/kg b.w. GT,b.i.d., oral at 08.00 AM and 04.00 PM. Group Insulin + GT (N = 5): 2 IUlong acting insulin b.i.d., SC + 60 mg/kg b.w. GT, b.i.d., oral at 08.00AM and 04.00 PM.

The single or combined oral doses per kg b.w. were solved in 5 mL 1%methylcellulose in saline.

The study was started with the 12 weeks old Zucker (fa/fa) rats. At thebeginning of the study a first OGTT was performed (dose: 2 g glucose/kgbody weight (b.w.); administration volume: 5 mL/kg of a 40% solution; B.Braun Melsungen, Melsungen, Germany) after a 16 h fast and an acutemedication. The glucose was administered via a feeding tube (15 g, 75mm; Fine Science Tools, Heidelberg, Germany). The group relevant drugswill be given as shown below:

Group characterization and medication for the first and second OGTTAnimals Group (N) Drug/Dose Comments CO 5 Placebo, 5 ml/kg b.w. 0.5mL/100 g b.w. 1% methylcellulose in saline at −5 min GT 5 GT, 60 mg/kgb.w. 60 mg GT solved in 5 mL 1% methylcellulose in saline, 0.5 mL/ 100 gb.w at −5 min Rosiglitazone + GT 5 Rosiglitazone, 3 mg/kg 3 mgRosiglitazone solved in 2.5 ml b.w. + P93/01, 60 mg/kg 1%methylcellulose in saline, b.w. 0.250 ml/100 g b.w. at −30 min; 60 mg GTsolved in 2.5 mL 1% methyosaline, 0.250 mL/100 g b.w at −5 minAcarbose + GT 5 No Acarbose preload 60 mg GT solved in 5 mL 1% beforeOGTT; GT, 60 mg/kg methylcellulose in saline, 0.5 mL/ b.w. 100 g b.w. at−5 min Glibenclamide + GT 5 Glibenclamide, 5 mg/kg 5 mg Glibenclamidesolved in 2.5 ml b.w. + GT, 60 mg/kg b.w. 1% methylcellulose in saline,0.250 mL/100 g b.w. at −40 min + 60 mg GT solved in 2.5 mL 1% methylcellulose in saline, 0.250 mL/ 100 g b.w. at −5 min Insulin + GT 5Insulin, 2 IU SC + GT; 60 mg/kg 2 IU Insulin SC Actrapid + 60 mg b.w. GTsolved in 5 mL 1% methylcellulose in saline, 0.5 mL/ 100 g b.w. at −5min.

Blood samples were taken from tail veins to measure blood glucose andserum insulin at −15, ±0 min, 15, 30, 60, 90, 120 and 180 min (thelatter time without insulin samples).

After first OGTT the animals in the groups was administered daily withthe respective drugs at 08:00 AM and at 04:00 PM, respectively:

During the two weeks of medication morning blood glucose was measuredbefore the 08:00 AM medication at Monday, Wednesday and Friday.

Food and water intake was determined every day during the time ofmedication.

All animals were weighted three times per week at 7:30 AM.

A second OGTT was performed after two weeks of medication (Day 15). Thefood will be withdrawn at 04:00 PM the day before (16 h fast). The OGTTwas be performed with the pre-medication to defined times and oralglucose loading at ±0 min. Blood samples were taken from tail veins tomeasure blood glucose and serum insulin at −15, ±0 min, 15, 30, 60, 90,120 and 180 min (the latter time without insulin samples).

After a wash-out period of one week a last OGTT was performed (>day 22).The food had been withdrawn at 04:00 PM the day before (16 h fast). TheOGTT was performed with administration of placebo to all groups (thismeans 1% methylcellulose and SC saline, respectively) to the definedtimes and oral glucose loading at ±0 min. Blood samples were taken fromtail veins to measure blood glucose and serum insulin at −15, ±0 min,15, 30, 60, 90, 120 and 180 min (the latter time without insulinsamples).

Glycated hemoglobin was measured before (Day −5) and after twice dailymedication for >two weeks (Day 18).

Measurements:

Glucose-For determination of glucose 20 μL blood was collected at −15,±0 min (before OGTT) and 15, 30, 60, 90, 120 and 180 min post OGTT.

Insulin-Insulin concentrations wer assayed by the antibody RIA method(Linco Research, Inc. St. Charles, Mo., USA).

Glycated hemoglobin-Percentage of glycated hemoglobin A (HbA1c) wasestimated with the “DCA 2000R Hämoglobin A1c-Reagenz kit” (Bayer VitalGmbH, Fernwald, Germany).

Body weight-The body weight was measured using a platform balance(Scaltec, Heiligenstadt, Germany).

Mixed venous blood samples from the tails were collected into 20 μLglass capillaries, which will be placed in standard tubes filled with 1ml solution for hemolysis (blood glucose measurement) and in sampletubes for serum insulin (50 μL blood). Blood samples in sample tubeswere centrifuged immediately (12.000 rpm for 2 min) and serum forinsulin analysis will be stored at −20° C. until analysis. Blood samplesare labeled with Protocol Number, Date of sampling, Time of sampling,Animal Number and Type of sample.

Results:

First OGTT under medication was performed at the beginning of the study.Animals of all experimental groups showed no differences in theirglucose tolerance after glucose load of 2 g/kg glucose. The same resultwas obtained regarding the baseline corrected glucose AUC_(1-180 min).Baseline was set as glucose value at time t=0.

Blood glucose during OGTT-2 after 14 days subchronic medication:

14 days subchronic treatment of fatty Zucker rats (fa/fa) has resultedin an improved glucose tolerance in all treatment groups versus Control.FIG. 7 shows the baseline corrected Area under the glucose-time-curveafter glucose load of 2 g/kg glucose on day 14.

In conclusion subchronic administration of glutaminyl thiazolidine aloneor in combination with Rosiglitazone, Glibenclamide, Acarbose or Insulinled to improved glucose tolerance.

1-26. (canceled)
 27. A method for glycaemic control in a mammal, such asa human, which method comprises administering an effective amount ofglutaminyl thiazolidine or glutaminyl pyrrolidine, or a pharmaceuticallyacceptable salt thereof, and another antidiabetic agent, to a mammal inneed thereof.
 28. A method for the treatment of diabetes mellitus andconditions associated with diabetes mellitus, the prediabetic stateand/or obesity in a mammal, which method comprises administering aneffective amount of glutaminyl thiazolidine or glutaminyl pyrrolidine,or a pharmaceutically acceptable salt thereof, and another antidiabeticagent, to a mammal in need thereof.
 29. The use according to claim 27 or28 for the treatment of Type 2 diabetes.
 30. The according to claim 27or 28 wherein the glutaminyl thiazolidine or glutaminyl pyrrolidine, ora pharmaceutically acceptable salt thereof, and the other antidiabeticagent are co-administered or administered sequentially or separately.31. The use according to claim 27 or 28 wherein the glutaminylthiazolidine or glutaminyl pyrrolidine, or a pharmaceutically acceptablesalt thereof, and the other antidiabetic agent are administered orally.32. The use according to claim 27 or 28 wherein the glutaminylthiazolidine or glutaminyl pyrrolidine, or a pharmaceutically acceptablesalt thereof, is glutaminyl thiazolidine hydrochloride.
 33. The useaccording to claim 27 or 28 wherein the other antidiabetic agent isselected from an alpha glucosidase inhibitor, a biguanide, an insulinsecretagogue or an insulin sensitiser.
 34. The use according to claim33, wherein the alpha glucosidase inhibitor is selected from acarbose,emiglitate, miglitol and voglibose.
 35. The use according to claim 34,wherein the alpha glucosidase inhibitor is acarbose.
 36. The useaccording to claim 33, wherein the biguanide is selected from metformin,buformin and phenformin.
 37. The use according to claim 36, wherein thebiguanide is metformin.
 38. The use according to claim 33 wherein theinsulin secretagogue is selected from glibenclamide, glipizide,gliclazide, glimepiride, tolazamide and tolbutamide, acetohexamide,carbutamide, chlorpropamide, glibomuride, gliquidone, glisentide,glisolamide, glisoxepide, glyclopyamide, glycylamide, glipentiderepaglinide and nateglinide.
 39. The use according to claim 33, whereinthe insulin sensitiser is a PPARy agonist insulin sensitiser.
 40. Theuse according to claim 33, wherein the insulin sensitiser is selectedfrom troglitazone, ciglitazone, pioglitazone, englitazone androsiglitazone.
 41. A method for the treatment of diabetes mellitus andconditions associated with diabetes mellitus, the prediabetic stateand/or obesity in a mammal, which method comprises administering aneffective amount of glutaminyl thiazolidine hydrochloride and metformin,to a mammal in need thereof.
 42. A pharmaceutical composition comprisingglutaminyl thiazolidine or glutaminyl pyrrolidine, or a pharmaceuticallyacceptable salt thereof, and another antidiabetic agent, and apharmaceutically acceptable carrier.
 43. A pharmaceutical compositionaccording to claim 42 wherein the glutaminyl thiazolidine or glutaminylpyrrolidine, or a pharmaceutically acceptable salt thereof, isglutaminyl thiazolidine hydrochloride.
 44. A pharmaceutical compositionaccording to claim 42 or 43 wherein the other antidiabetic agent is asdefined in claim
 33. 45. A pharmaceutical composition comprisingglutaminyl thiazolidine hydrochloride and metformin.